Bovine ovarian superovulation is the current state-of-the-art method employed worldwide to maintain and improve milk and beef production through genetic selection of the best quality breeders and best quality embryos for transfer. These embryos may either be transferred fresh or frozen both to local recipient cows as well as conveniently shipped to any international locale. The current international market for superovulation is about $20 million per year but because of worldwide hunger, population growth and demand it has been predicted that this total market may exceed $200 million in the next decade. There are many limitations of current bovine ovarian superovulation and wide variations of the number of viable embryos recovered. By far the greatest factors in the variability of different lots are LH hormone contamination and microheterogeneity in currently used impure extracts of porcine pituitaries. Use of bovine pituitary-derived FSH has been banned by FDA and other national regulatory agencies because of possible contamination by fatal bovine spongiform encephalopathy which causes a related fatal disease in humans after ingestion. Several European countries have additionally banned the use of any veterinary pituitary products from any species, as had occurred decades earlier by all regulatory agencies when a similar fatal prion-caused encephalopathy had been discovered in humans from hormones derived from human pituitaries. In the current proposal the PIs will: 1. Develop novel high affinity recombinant bFSH superagonist analogs by minimal 4-5 arginine and/or lysine site-directed mutagenesis to produce a more potent and efficacious molecule with possible delayed absorption to increase duration of action; for selected bFSH superagonists also engineer a novel minimal length amino acid insert containing one or two complex carbohydrate neoglycosylation sites to increase half life to further increase duration of action and produce a single injection analog without reducing increased superagonist potency/efficacy plus reduced potential for immunogenicity; initially express these novel analogs by transient transfection using a previously optimized, novel, high expression & low cost production method. 2. Partially purify selected 4-6 analogs expressed by transient transfection and characterize in vitro by previously validated bFSH analog ELISA immunoassay, robust in vitro bioassay, SDS-PAGE electrophoresis and isoelectric focusing gel analysis. 3. Select the 3-5 best bFSH analog candidates to analyze in vivo by pK studies in mice and/or rats as well as the classic Steelman-Pohley FSH bioassay of ovarian weight gain plus histological analysis of antral follicle number and size. 4. Based on in vivo analysis, select the best 2-3 candidate analogs to construct dicistronic expression vectors, and initiate amplification of analogs expression in CHO-DG44 cells in preparation for Phase 2 large-scale production, purification and superovulation studies in cattle. This completely novel recombinant bFSH analog protected by international patents until 2032 & without a known recombinant competing product under development in the US should increase the efficacy, convenience & safety of bovine superovulation at much reduced cost. PUBLIC HEALTH RELEVANCE: Production of many eggs and embryos by hormone stimulation in female cows, termed superovulation, is the current state-of-the-art method to maintain and improve milk and beef production in response to worldwide hunger and population growth. There are many limitations of the current impure stimulating hormone used, called FSH, derived from pig pituitaries, including possible contamination by a protein infectious agent which causes a 100% fatal brain disease in humans after eating such infected meat. The two investigators have invented a completely new and more active DNA - derived form of cow-like FSH which cannot contain any infectious agent & should increase the efficacy, convenience & safety of cow superovulation at much reduced cost.